Holy Basil leaf extract decreases tumorigenicity and metastasis of aggressive human pancreatic cancer cells in vitro and in vivo: Potential role in therapy
Introduction
Pancreatic cancer (PC) has a dismal prognosis with a median 5-year survival of about 5% [1]. This is in large part due to the intrinsic resistance of PC cells to chemo and radiation therapy [2], [3]. Thus, there has been a concerted effort towards the development of newer drugs that can overcome this inherent resistance. Plant-derived chemicals (phytochemicals) have emerged as a potential source of novel anticancer compounds. Several natural products including curcumin [4], fisetin [5] and thymoquinone [6] have been demonstrated to enhance the sensitivity of PC cells to chemotherapeutic agents. Although initially promoted as chemopreventive drugs, they have also shown significant pro-apoptotic, anti-proliferative and anti-metastatic effects on cancer cells, prompting a call for their introduction as therapeutic agents.
Ocimum sanctum (commonly known as “Holy Basil”) is a medicinal herb found in the semitropical and tropical parts of India. It has been used for thousands of years in the Ayurvedic and Siddha systems of medicine to treat diverse ailments including infections, skin and liver disorders and as an antidote for snake and scorpion bites [7]. It has been used as an anti-inflammatory, immunomodulatory, anti-infective, anti-stress, antipyretic, antitussive, anti-diabetic [8], cardioprotective, neuroprotective and hepatoprotective agent [9], [10]. Although every part of the plant has been suggested to have therapeutic uses, the leaves (and leaf extracts) have been most extensively studied. The leaves of O. sanctum are the source of an essential oil which has numerous medicinal properties. Both ethanolic and essential oil basil extracts have previously been shown to have antioxidant effects [10], [11], [12], [13], [14], [15]. Ethanolic extracts have been shown to promote epithelialization of wounds and to counteract the healing suppressant effect of dexamethasone in albino rats [16]. Eye drops containing O. sanctum leaf extract protected against ferric chloride-induced lipid peroxidation and exhibited significant antibacterial and antifungal activity [13]. Another study has shown that essential basil oil fed to male Wistar rats significantly decreased the levels of serum lipids [14], while supplementation of fresh basil leaves (2 g/kg) daily for 30 days significantly lowered blood glucose and peroxidized lipid levels [17].
Few studies have also demonstrated the potential of O. sanctum as an anti-tumor agent [18]. In a comparison of the cytotoxic activity of essential oils from 17 Thai medicinal plants, O. sanctum was the most effective in inhibiting the proliferation of human oral squamous cell carcinoma (KB) and mouse leukemia cells (P388) in vitro [19]. Other studies have shown that its ethanolic extracts exhibited a cytotoxic effect against A549 lung cancer cells, cleaved the pro-apoptotic molecule poly-(ADP-ribose) polymerase (PARP), promoted the release of cytochrome C, and increased the activity of caspases 3 and 9 and the ratio of Bax/Bcl-2 [20]. It also decreased the rate of proliferation as evidenced by a decrease in the percentage of cells in the G2/M phase. The ethanolic extracts of O. sanctum have also inhibited the invasion of murine Lewis lung cancer (LLC) cells in vitro associated with a decrease in the activity of matrix metalloproteinase-9 (MMP9) [20]. In vivo, it significantly reduced the number of metastatic lung nodules following injection of LLC cells through the tail vein [21]. Overall, these studies suggest that extract from the leaves of the plant can induce apoptosis, inhibit cell cycle progression and prevent metastasis.
In the present study, we investigated whether the essential oil or extracts prepared from commercially available dried leaves of O. sanctum could inhibit proliferation, survival and metastasis of PC cells. The results of our study reveal that both ethanolic extracts (EEOL) and the essential oil of O. sanctum leaves (EOOS) significantly inhibit the aggressiveness of PC cells and inhibit the growth of orthotopically implanted PC cells. Overall, our study is the first to suggest a potential role for O. sanctum in the therapy of PC.
Section snippets
Preparation of ethanolic extracts of O. sanctum leaves (EEOLs)
We purchased capsules containing powdered dried leaves of O. sanctum from four vendors in the United States: New Chapter (NC), (New Chapter Inc., Bloomingdale, IL, USA), Club Natural (CN), (Club Natural Inc., Irvine, CA, USA), Superior Herbs (SH), (Swanson Health Products, Fargo, ND, USA) and Morpheme (Morph), (Morpheme Remedies Pvt. Ltd., Panchkula, Haryana, India). We also purchased the essential oil of O. sanctum leaves (EOOS) from Now Foods, (Bloomingdale, IL, USA).
Ethanolic extracts of O.
Determining the optimum dose of ethanolic extracts of O. sanctum leaves (EEOLs) and essential oil of O. sanctum leaves (EOOS) to inhibit the proliferation of pancreatic cancer cells in vitro
We first sought to determine the optimum dose of EEOLs and EOOS for in vitro treatment of PC cells from various sources. For this, AsPC-1, MiaPaCa, CD18/HPAF, and Capan-1 PC cells were treated with EEOL (2-fold serial dilutions between 2 mg/ml and 1 μg/ml) or EOOS (2-fold serial dilutions between 2% and 0.001% v/v) for 48 h. Cell viability was determined by MTT assay and the IC-50 was calculated as described in Section 2. As shown in Fig. 1A, the half maximal inhibitory concentration (IC-50) of the
Discussions
O. sanctum, also known as Ocimum tenifluorum or Holy Basil, an herb belonging to the family Lamiaceae, is a well-known medicinal plant in use for several thousands of years in the traditional Indian system of medicine. While most studies have focused on the therapeutic effects of O. sanctum leaf extracts on benign diseases, studies of its anti-cancer effects are sparse.
To our knowledge, there are no published studies of the effect of basil extracts in PC cells. We observed that both EOOS and
Conflicts of interest
There are no potential conflicts of interest involved with this work.
Acknowledgments
The invaluable technical support from Kavita Mallya is greatly appreciated. We also thank Janice A. Tayor and James R. Talaska of the Confocal Laser Scanning Microscope Core Facility at UNMC, Victoria B. Smith and Megan Michalak of the UNMC Cell Analysis Core Facility, and the Eppley Cancer Center for their support of the core facilities. The authors of this work are supported by Grants from the National Institutes of Health: NIH-NCI Cancer Biology Training Grant UNMC T32CA009476 (MPT, JS), VA
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These authors equally contributed to the work.